FAQs

A. Pharmacy Teaching
See Moodle units

B. Microscope Suite 'Emergencies'
B1. The incubators are sounding an alarm: low CO2. How do I change the cylinder?
If James is around, contact him. Otherwise, do the following:
Go into the Imaging Suite (3.18). The CO2 cylinders are on the left as you walk in. High above the middle of the two CO2 cylinders is a lever. Push it in the opposite direction (up or down) to switch in the other cylinder. No other action required.
B2. The hypoxic incubators are sounding an alarm: low N2/High O2. How do I change the cylinder?
If James is around, contact him. Otherwise, do the following:
Go into the Imaging Suite (3.18). The N2 cylinders are on the right as you walk in. Check that the only connected cylinder is reading zero on the left hand dial (usual cause). Turn off cylinder using rod-like spindle key already connected to cylinder (direction shown on edge of cylinder; clockwise). Once closed, disconnect metal tubing to cylinder using spanner on hexagon-screw - turn anticlockwise to disconnect. Locate a new cylinder; this will have a blue plastic top for you to rip off (a blue top already ripped off but balanced on top the cylinder mean that it is partly full - normally good enough for overnight). Place metal tubing onto new cylinder and use spanner on hexagon-screw (clockwise) to tighten it (hard as you can). Then open the cylinder using the rod-like spindle key (anticlockwise to open); turn this key in about 4 or 5 half-turns to fully open. You will hear a hiss. No other action required.
B3. I'm getting an error message when trying to open Volocity. What shall I do?
The Volocity license server has run out of available licenses - too many users currently on the system.
If you are working at home, there's not much you can do - try going back on later.
If near the Imaging Suite, observe the high number of users or the license server PC (right hand side when entering Imaging Suite). Only note the 0 available licenses, usually under the Visualisation licenses category. Please reboot the license server PC. This won't affect anybody else (since users on Volocity make an initial connection with the license server PC and do not need to remain connected). Use username and password: '.\Volocity' and 'glioma' and once Windows has loaded, double click on the iExplore icon. The licence server web page will then load and operate automatically. Restart your Volocity program.
B4. The licence server seems to be down/has been switched off (perhaps after a power failure) - what shall I do, as Volocity will not load?
Please reboot the license server PC (right hand side when entering Microscopy suite. Use username and password: '.\Volocity' and 'glioma' and once Windows has loaded, double click on the iExplore icon. The licence server web page will then load and operate automatically. Restart your Volocity program.
B5. I can't get into Volocity. I get the error message: 'The command could not be completed because licenses are not available for all the packages in the selected license server configuration (2616).
All the licenses are currently being used. Make sure you are not using the 'Acquisition' set up if you are just doing image processing and not connected to a microscope. Other options are to log in to Volocity again later or better still, have a look to see who is using Volocity (click here and checking to see what licenses are available/who is using Volocity (if somebody is not using the acquisition modes, you might be able to arrange something. The final option, which always works but only when you're in the Imagaing Suite, is to reboot the license server - which does not affect existing users nor projects currently using Volocity - for this, see B4 above.

C. Microscopy/Fluorescence/Cell Tracking/Scratch Assays/Volocity
C1. I want to use one of the microscopes; is there a booking system?
Yes. Please contact James about what's required and then a booking will be made via Google Calendar.
C2. Could you tell me the name of the microscope that we used? / What instruments do you have?
Please contact James so that you can be added to the Advanced Microscopy moodle course that will give such details. You will need your UP number if you are a student.
C3. Do you have any inverted microscopes?
Yes, live cell imaging and the TIRF microscope are inverted. The latter cannot image well plates - only slides.
C4. What imaging modes/filter sets are available with live cell imaging?
Brightfield, phase imaging, DIC and fluorescence with filtersets 49 (DAPI,blue), 10 (FITC,green), 37 (GFP,green), 20 (Rhod,red) and 0 (Tex Red,red). The excitation and emission wavelengths and other details are summarised on the Zeiss website here.
C5. Apart from the live cell imaging, are there any other microscopes that can do fluorescence and what filter sets do they have?
There are 2 other main microscopes capable of fluorescence on the 3rd floor of SMB (not including the confocal instruments). These are the TIRF microscope (a Zeiss AxioVert 200M; inverted microscope; Room 3.18) which does not have to be run in "TIRF-mode" and the Zeiss AxioImager.Z1 (not inverted microscope; 'Charlie's Challenge' Room 3.38 within Room 3.37).
The TIRF microscope has a 45 (Alexa568,red) and 49 (DAPI,blue) filter sets and a 488 nm/FITC [38] type filter (specially designed for TIRF). The camera on the TIRF is an AxioCam HSm (monochrome, high speed and therefore slightly lower res than normal). The objectives are 10x, 20x, 40x (all air).
The AxioImager.Z1 (Charlie's Challenge Room) has filter sets 20 (Rod,red), 38 (FITC,green) and 49 (DAPI,blue), and 50 (far red), with a Hamamatsu C4742-95 monochrome camera (a colour QImaging camera for brightfield is also possible on this microscope). The objectives are 2.5x, 5x, 10x, 20x, 40x (all air) and 40x oil. The TIRF microscope can only use microscope slides, but the AxioImager.Z1 (Charlie's Challenge) can use well plates despite it not being an inverted microscope. Well plates are best used on live cell imaging microscope (Zeiss AxioVert 200M).
The excitation and emission wavelengths and other details are summarised on the Zeiss website here.
There is a second live cell imaging instrument (EVOS microscope) but this is only funded for Neurooncology use. Further details for the EVOS microscope are on the Advanced Microscopy moodle page.
C6. What plates can be used for live cell imaging?
Any standard size plate from any manufacturer can now be used. Previously, only Falcon 24 well plates could be used, but the new stage allows for more variation. You may like to enquire with James if you are not sure. Please ask your line mamanger if you do not have your own supply of well plates.
C7. Can I use Volocity on my own PC?
Yes, both at work and at home. Please see ask James to add you to the Advanced Microscopy moodle site. Info for Volocity installation and protocols can be found from there.
C8. Is there a manual for Volocity?
This 536 page document(!) can be downloaded here. This is also available from the Advanced Microscopy moodle site - ask James to add you.
C9. Are there any operating procedures for the microscopes/Volocity software?
These are available from the Advanced Microscopy moodle site - ask James to add you.
C10. Have you got the contact details for outside help with Volocity software?
Help from Perkin-Elmer can be found here.
The tel number has now changed to 0800 896046.
C11. Can I process a Zeiss microscope image (.zvi; not acquired from Volocity) on my own PC?
If the image was acquired using AxioVision (and NOT Volocity, e.g., when using the TIRF instrument), then a "light" version of AxioVision (AxioVision LE) can be downloaded from here, although installing it on a networked computer will only be possible if you have admin rights (most people don't, see next Q).
C12. How do I get access rights?
PhD students can get access rights, please ring IS, but unfortunately this is not possible for project and Erasmus students. See James.
C13. Is there a microscope with a colour camera (on the 3rd floor)?
There is. It's the QImaging colour camera located on the Zeiss AxioImager.Z1 in Room 3.38 (Charlie's Challenge; in Room 3.37). Within Volocity, under Source, just change the Hamamatsu camera (greyscale) to the QImaing colour camera. There is now no need to connect/disconnect firewire cables (leave them attached). Please let James know if you have any queries/difficulties changing/using these cameras.
C14. I want to use SEM. Who should I contact?
Please contact Christine Hughes in Biology (King Henry Building) for all EM/SEM/TEM queries.
C15. I want to use the confocal microscope(s). How should I contact?
Please contact Jill Rice.
C16. Are the confocal microscopes networked?
No. Use a CD.

D. AFM
D1. Can you recommend any good books/reading on AFM, ideally from the library?
These are available from the Advanced Microscopy moodle site - ask James to add you.
The following books are in the library: Eaton, P., West, P. Atomic force microscopy, OUP, 2010; Morris, V.J., Kirby, A.R., Gunni, A.P. Atomic force microscopy for biologists, Imperial College Press, London, 1999; Braga, P.C. Atomic force microscopy: biomedical methods and applications, Humana Press, Totowa, NJ, 2004. There are a few others shelved at 502.8. There are also many good review papers - see web of science or sciencedirect - and websites: AFMHelp.com
D2. Could you tell me the name of the microscope that we used?
This info is available from the Advanced Microscopy moodle site - ask James to add you.
D3. What is the difference between AFM and SPM/STM?
Atomic force microscopy is the second probe microscopy technique under the general heading scanning probe microscopy (SPM). AFM allows imaging of insulating materials and therefore is used more widely than the first technique, scanning tunnelling microscopy, which relies on monitoring a tunnelling current between a conducting tip and substrate. We can no longer perform STM at Portsmouth.

E. General/Admin
E1. How do I use EndNote or EndNote Web?
Recommend using EndNote Web where references are stored online. UoP Youtube video can be found here: Part 1 and Part 2.
E2. How do I use EndNote Web remotely/at home? Is it something to do with an Athens account?
Athens is no longer used: Shiobboleth is the new access management system... What you must do is Web cache your home computer so that it looks as if it's on-campus; instructions for doing this can be found at http://www.port.ac.uk/library/helpyourself/gettingstarted/webproxy/

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